Receptors for the Fc portion of immunoglobulins (Fc receptors) play a fundamental role in immune defense by controlling the interactions of antibodies with effector cells, thereby providing an important connection between humoral and cellular immunity. These receptors mediate cell activation by immune complex and/or antigen and are involved in the generation of both protective immune responses and the pathological inflammatory reactions associated with certain immune disorders, immune complex diseases and allergy. The high-affinity IgE receptor (FcepsilonRI) mediates the activation of mast cells (MC) and basophils and induces the release of various inflammatory mediators, while the low affinity IgG receptor (FcgammaRIII) is involved in mediating many of the cellular responses associated with the presence of IgG-containing immune complexes. Our laboratory has produced monoclonal antibodies (mAbs) with a capacity to inhibit FceRI-mediated MC activation to use them as tools to identify and characterize novel inhibitory mechanisms of FcR-mediated activation. Some of these mAbs are directed against two tetraspanins, CD81 and CD63. They are capable of inhibiting MC IgE-mediated degranulation in vitro and in vivo. Inhibition of IgE-mediated MC degranulation takes place without affecting earlier signals such as tyrosine kinase and calcium mobilization. Instead, anti-CD63 inhibits the adaptor Gab2, a central component of the degranulation pathway induced by FceRI aggregation. In addition, anti-CD63 inhibits MC adhesion specifically to some integrin substrates and exocytosis of MC adhering to these same substrates. Preliminary evidence suggests that anti-CD63 affects MC growth. Our central hypothesis is that membrane complexes containing CD63 and CD81 use the Gab2/PI3K pathway to regulate MC functions such as FcR-mediated degranulation, Beta-integren mediated adhesion and survival/proliferation. We propose experiments (1) to assess whether the anti-CD81 and anti- CD63 target the same pathway and whether both mAbs can act synergistically;(2) to identify signals upstream of the Gab2/PI3K pathway regulated by CD63 and CD81;(3) to explore whether the role of CD63 and CD81 extends to IgG-mediated and FcR-independent MC functions in vitro and in vivo;(4) to investigate the long term effects of the mAbs on MC survival and proliferation;(5) to identify the different functional regions of CD63 and CD81 that are involved in the regulation of MC functions. The answers to these questions will contribute greatly to our understanding of how these Fc receptors mediate inflammatory responses in the context of host defense and immunological diseases. Moreover, they will provide the proof of concept for the therapeutic use of these antibodies in the context of MC-associated diseases.